Fibronectin (Fn) is an abundant 450kDa mammalian glycoprotein found in a soluble form in body fluids and deposited as a fibrillar, insoluble form in the Extracellular Matrix (ECM) of most tissues. It plays critical roles in embryonic development, normal biology, and pathology. In addition, it appears that Fn is the favored host target for pathogenic microorganisms. To date, over 100 different microbial proteins have been reported to bind to Fn, however, most of these interactions have not been characterized and the biological consequences are usually not known. We hypothesize that the interaction of bacterial components and host Fn can have different biological consequences depending on the Fn domains targeted. We propose that the MSCRAMMs contain specific motifs that allow them to interact with different specific Fn domains thereby exploiting the various aspects of the biological activity of Fn. In support of these hypotheses we have shown that intrinsically disordered MSCRAMM segments and linear peptides can target the N-terminal domain (NTD) in Fn and that this is a signature for a host cell invasion process involving Fn and 1521 integrins. These sequence motifs were first found in MSCRAMMs from Staphylococcus aureus, Streptococcus pyogenes and Borrelia burgdorferi. In preliminary studies we have detected similar motifs in other Fn-binding MSCRAMMs and propose to determine their role in the ligand interaction. Many MSCRAMMs bind to multiple domains in Fn. The Gelatin Binding Domain (GBD) is targeted by several microbial proteins. We will explore the hypothesis that this interaction interferes with the documented motogenic activity of the GBD. Some MSCRAMMs target F3 modules and at least one of these BBK32 from B. burgdorferi, can induce the formation of superfibronectin (sFn). This process appears to involve an interaction of a linear sequence motif in the MSCRAMM with multiple Fn F3 modules. We will define this putative binding motif in BBK32 and determine if other Fn binding proteins contain the motif. In addition, we will explore the possibility that the F3 binding MSCRAMMs can regulate the interaction of Fn with 1921 and 1421 integrins. To examine different aspects of our general hypothesis we will characterize the interaction of Fn-binding motifs in MSCRAMMs with their ligands and determine the biological consequences of these interactions in vitro. In these experiments we will use a combination of biochemical, structural and cell biological methods. PUBLIC HEALTH RELEVANCE: Many infectious organisms express surface proteins that bind fibronectin (Fn). Fn is a human protein that is very important for human growth and development as well as in maintaining hemostasis. In addition, Fn plays critical roles in inflammation, cancer and wound healing. Many microorganisms require Fn-binding proteins to be fully infectious. We propose to characterize the microbe-Fn interaction in detail and to determine the molecular basis for the role of Fn-binding proteins in infectious disease.